Our laboratory is focused on determining the pathogenesis mechanism(s) of age related macular degeneration (AMD). AMD is a complex disease involving the retinal pigment epithelium (RPE) and mediated by the aging process as well as genetic and environmental factors. Thus, we have been searching for common age-related factors that may be adversely affecting the health of the RPE and that initiate before the macular pathology is clinically detectable. Research in atherosclerosis, also a complex age-related disease, suggests that LDL deposits and accompanying oxidized forms of cholesterols or oxysterols may be playing an important role in its pathogenesis. Oxysterols are known to have potent pharmacological effects on many different cell types, including inhibition of cholesterol synthesis, gene expression and apoptosis. The immune system is also known to play an important role in atherosclerosis because macrophages that phagocytize oxidized LDL become foam cells, which are part of the atherosclerotic plaques. Other investigators have demonstrated that the accumulation of LDL cholesterol as well as unesterified sterols in the choriocapillaris and Bruch's membrane seems to be a normal process in the aging human eye. LDL cholesterol is one of the major sources of oxidized cholesterol and its retention by Bruch's membrane and the choriocapillaris would offer an opportunity for further oxidation. This oxidized LDL may serve as a source of oxysterols that in turn adversely affects the health of the RPE. Thus, our hypothesis for the pathogenesis of AMD is that the gradual accumulation of LDL in Bruch's membrane causes the hyperoxidation of LDL, which in turn attracts the immune system, mainly macrophages. The degradation of LDL by the macrophages as well as by endogenously released lipases and proteases causes the release of oxysterols, which in turn cause cytotoxic effects on the RPE and surrounding choroidal endothelium. Macrophages also die as a result of ingesting oxidized LDL and thus further contributing to debris accumulation and cytotoxicity. Symptoms of AMD begin when the functionality of the RPE begins to degrade and is usually manifested with the formation of drusen deposits. These deposits are possibly caused by debris left from the dead immune cells, lack of proper waste processing by the RPE, and the fouling of Bruch's membrane by LDL and related components. We are presently involved in investigating the mechanisms of oxysterol cytotoxicity in cultured RPE cells. We are accomplishing this by investigating the oxysterol binding proteins (OSBPs) and other oxysterol related proteins. The OSBPs are a family of proteins that bind oxysterols and may be involved in mediating their pharmacological effects. Our laboratory has cloned twelve different OSBPs and determined their primary structure and tissue expression. We are in the process of determining their intracellular targeting and differential binding activity by making green fluorescent protein fusion constructs containing different portions of the OSBP molecules. Stably transfected cell lines over-expressing different OSBPs are also being constructed to determine how over-expression affects cytotoxicity.